The Drosophila central nervous system (CNS) is composed of hundreds of neurons, each expressing a unique combination of neurotransmitters, ion channels, receptors, and cell surface molecule -- with each neuron forming numerous stereotyped contacts with other neurons or muscles. How is this spectacular neuronal diversity generated? Our long term goals are to identify the cellular and molecular mechanisms used to generate neuronal diversity in the Drosophila CNS, and determine whether these mechanisms are conserved in more complex organisms. Drosophila is an ideal organism for investigating CNS development: neurogenesis is relatively simple, with cellular diversity being generated in two steps. First, positional cues in the neuroectoderm control the formation of 25 unique neuronal precursor cells (neuroblasts); second, each neuroblast goes through an invariant cell lineage to generate an average of five ganglion mother cells (GMCs) which each produce a pair of neurons. Each of these two steps contributes to the diversity of cell fate in the CNS, and each step can be studied independently. The proposed research uses several recent technical advanced to investigate both neuroblast and neuronal determination. To understand how positional cues control neuroblast determination, we will make use of recently developed molecular markers that allow each neuroblasts to be uniquely identified, and assay for mutations that alter expression of these markers. In particular, we will focus on the role of the segment polarity gene wingless in the non-autonomous determination of the identified NB4-2 (Specific Aim I). We will also focus on the role of the 5953 gene, which is expressed in the identified NB4-2 in response to the wg positional cue, and is apparently required for NB4-2 determination. To characterize genes controlling lineage-based neuronal determination, we will look for specific alterations in neuroblast cell lineages using a novel cell marking technique to label single identified neuroblast and all of its neuronal progeny. In particular, we will investigate the role of ming, seven-up and prospero genes in the specification of cell fate during the lineage of the identified NB1-1, NB4-2 and NB7-4 (Specific Aim II). In addition, we will use molecular markers for specific neuroblast "sublineages" and mutations in cell cycle control genes to investigate the relationship between transition through the cell cycle and the sequential specification of GMC fate during identified neuroblast lineages (Specific Aim III). We anticipate rapid progress in identifying and characterizing genes controlling the generation of neuronal diversity in the Drosophila CNS due to the relative simplicity of the nervous system molecular markers for individual precursor cells, powerful new techniques for identifying and cloning CNS-expressed genes, and a newly developed cell lineage method that allows wild-type and mutant NB lineages to be directly compared at the level of the axonal projections of every neuron in the linage.